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Binding polypeptides with restricted diversity sequences

a polypeptide and diversity technology, applied in the field of variable cdrs, can solve the problems of inability to apply systematic and quantitative methods, limited diversity of these libraries, and inability to meet the needs of patients,

Inactive Publication Date: 2007-10-11
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] The present invention provides simplified and flexible methods of generating polypeptides comprising variant CDRs that comprise sequences with restricted diversity yet retain target antigen binding capability. Unlike conventional methods that are based on the proposition that adequate diversity of target binders can be generated only if a particular CDR(s), or all CDRs are diversified, and unlike conventional notions that adequate diversity is dependent upon the broadest range of amino acid substitutions (generally by substitution using all or most of the 20 amino acids), the invention provides methods capable of generating high quality target binders that are not necessarily dependent upon diversifying a particular CDR(s) or a particular number of CDRs of a reference polypeptide or source antibody. The invention is based, at least in part, on the surprising and unexpected finding that highly diverse libraries of high quality comprising functional polypeptides capable of binding target antigens can be generated by diversifying a minimal number of amino acid positions with a highly restricted number of amino acid residues. Methods of the invention are rapid, convenient and flexible, based on using restricted codon sets that encode a low number of amino acids. The restricted sequence diversity, and thus generally smaller size of the populations (e.g., libraries) of polypeptides generated by methods of the invention allows for further diversification of these populations, where necessary or desired. This is an advantage generally not provided by conventional methods. Candidate binder polypeptides generated by the invention possess high-quality target binding characteristics and have structural characteristics that provide for high yield of production in cell culture. The invention provides methods for generating these binder polypeptides, methods for using these polypeptides, and compositions comprising the same.

Problems solved by technology

However, many of these libraries have limited diversity.
The size of the library is decreased by inefficiency of production due to improper folding of the antibody or antigen binding protein and the presence of stop codons.
However, these attempts have had varying success and have not been applied in a systematic and quantitative manner.
Creating diversity in the CDR regions while minimizing the number of amino acid changes has been a challenge.
However, these analyses have generally been limited in scope and nature, and substantial skepticism and questions remain regarding the feasibility of generating polypeptides having complex functions using a restricted set of amino acid types.

Method used

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  • Binding polypeptides with restricted diversity sequences
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  • Binding polypeptides with restricted diversity sequences

Examples

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example 1

Construction of Phage-Displayed Fab Libraries with CDR Residues Enriched in Tyr or Ser

[0686] Phage-displayed Fab libraries were constructed using the “Fab-C” phagemid vector that resulted in the display of bivalent Fab moieties dimerized by a free cysteine inserted between the Fab heavy chain and the C-terminal domain of the gene-3 minor coat protein (P3C). This vector was constructed as described in U.S. Patent Application Publication No. US20050119455 and in Lee et al., J. Immunol. Meth. 284: 119-132 (2004). The vector (schematically illustrated in FIG. 5) comprises the humanized antibody 4D5 variable domains under the control of the IPTG-inducible Ptac promoter. The humanized antibody 4D5 has mostly human consensus sequence framework regions in the heavy and light chains, and CDR regions from a mouse monoclonal antibody specific for Her-2. Methods of making the anti-Her-2 antibody and the identity of the variable domain sequences are provided in U.S. Pat. Nos. 5,821,337 and 6,05...

example 2

Selection of Specific Antibodies from the Naïve Libraries YS-C and YS-D

[0692] Phage from library YS-C or YS-D (see Example 1) were cycled through rounds of binding selection to enrich for clones binding to human VEGF. The binding selections were conducted using previously described methods (Sidhu et al., supra).

[0693] NUNC 96-well Maxisorp immunoplates were coated overnight at 4° C. with 5 μg / mL human VEGF and blocked for 2 h with a solution of PBT (phosphate buffered saline additionally containing 0.2% BSA and 0.05% Tween-20) (Sigma). After overnight growth at 37° C., phage were concentrated by precipitation with PEG / NaCl and resuspended in PBT, as described previously (Sidhu et al., supra). Phage solutions (about 1012 phage / mL) were added to the coated immunoplates. Following a two hour incubation to permit phage binding, the plates were washed ten times with PBT. Bound phage were eluted with 0.1 M HCl for 10 minutes and the eluant was neutralized with 1.0 M Tris base. Eluted ph...

example 3

Construction of Phage-Displayed Fab Libraries with CDR Residues Enriched in Tyr, Ser, Gly, and Arg

[0698] Phage-displayed Fab libraries were constructed using a phagemid vector, Fab-C, that resulted in the display of bivalent Fab moieties dimerized by a free cysteine inserted between the Fab heavy chain and the C-terminal domain of the gene-3 minor coat protein (P3C), as previously described in Example 1.

[0699] Four libraries were constructed: YSGR-A, YSGR-B, YSGR-C, and YSGR-D. The libraries were constructed with randomized residues in all three heavy chain CDRs and light chain CDR3. Each library was randomized at positions 91-94 and 96 of CDRL3, positions 28 and 30-33 of CDRH1, positions 50, 52-54, 56, and 58 of CDRH2, and positions 95-100, 100a, 100b, and 100c of CDRH3. The type and ratio of the amino acids allowed at each of the randomized positions is described in FIG. 11. In addition, the length of CDRH3 was varied by using oligonucleotides that replaced the seven wild-type c...

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Abstract

The invention provides variant CDRs comprising highly restricted amino acid sequence diversity. These polypeptides provide a flexible and simple source of sequence diversity that can be used as a source for identifying novel antigen binding polypeptides. The invention also provides these polypeptides as fusion polypeptides to heterologous polypeptides such as at least a portion of phage or viral coat proteins, tags and linkers. Libraries comprising a plurality of these polypeptides are also provided. In addition, methods of and compositions for generating and using these polypeptides and libraries are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a nonprovisional application which claims priority to U.S. Ser. No. 60 / 742,184 filed Dec. 2, 2005 and U.S. Ser. No. 60 / 805,553 filed Jun. 22, 2006, all of which applications are incorporated by reference herein.FIELD OF THE INVENTION [0002] The invention generally relates to variant CDRs diversified using highly limited amino acid repertoires, and libraries comprising a plurality of such sequences. The invention also relates to fusion polypeptides comprising these variant CDRs. The invention also relates to methods and compositions useful for identifying novel binding polypeptides that can be used therapeutically or as reagents. BACKGROUND [0003] Phage display technology has provided a powerful tool for generating and selecting novel proteins that bind to a ligand, such as an antigen. Using the techniques of phage display allows the generation of large libraries of protein variants that can be rapidly sorted for thos...

Claims

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Application Information

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IPC IPC(8): C07K16/18A61K39/395C07H21/04C07K16/46C12N5/16G01N33/563C12N15/63C07K1/00C07K14/00
CPCC07K14/70503C07K16/32C07K2317/92C07K2317/55C07K2317/565C07K2317/24
Inventor BIRTALAN, SARA C.FELLOUSE, FREDERICSIDHU, SACHDEV S.
Owner GENENTECH INC
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